Current State of Knowledge Regarding WHO Critical Priority Pathogens: Mechanisms of Resistance and Proposed Solutions through Candidates Such as Essential Oils

The rise of multidrug-resistant (MDR) pathogens has become a global health threat and an economic burden in providing adequate and effective treatment for many infections. This large-scale concern has emerged mainly due to mishandling of antibiotics (ABs) and has resulted in the rapid expansion of antimicrobial resistance (AMR). Nowadays, there is an urgent need for more potent, non-toxic and effective antimicrobial agents against MDR strains. In this regard, clinicians, pharmacists, microbiologists and the entire scientific community are encouraged to find alternative solutions in treating infectious diseases cause by these strains. In its “10 global issues to track in 2021”, the World Health Organization (WHO) has made fighting drug resistance a priority. It has also issued a list of bacteria that are in urgent need for new ABs. Despite all available resources, researchers are unable to keep the pace of finding novel ABs in the face of emerging MDR strains. Traditional methods are increasingly becoming ineffective, so new approaches need to be considered. In this regard, the general tendency of turning towards natural alternatives has reinforced the interest in essential oils (EOs) as potent antimicrobial agents. Our present article aims to first review the main pathogens classified by WHO as critical in terms of current AMR. The next objective is to summarize the most important and up-to-date aspects of resistance mechanisms to classical antibiotic therapy and to compare them with the latest findings regarding the efficacy of alternative essential oil therapy.


Introduction
Antimicrobial resistance (AMR) is a process that occurs naturally and has been known for more than 50 years, when Staphylococcus aureus began to develop penicillin resistance [1]. Nowadays, AMR has become a public health emergency, mainly due to inappropriate use of in human and animal health and increasing investment in new drugs and vaccines, as part of the main strategies to combat the spread of resistant restrains [16].
Since 2017, nine new antibacterial active ingredients were approved by the Food and Drug Administration (delafloxacin, eravacycline, meropenem-vaborbactam, plazomicin, omadacycline, cefiderocol, pretomanid, relebactam-imipenem/cilastatin and lefamulin) and one vaccine for tuberculosis [17]. Nearly half of the recently approved ABs act against carbapenem-resistant Enterobacterales, Oxacillinase-48-producing Enterobacterales and β-lactamase-producing Enterobacterales [17]. A total of 32 antibiotics act on the pathogens mentioned in the WHO's priority pathogens list [17]. However, new treatment options for carbapenem-resistant A. baumannii and carbapenem-resistant P. aeruginosa are still lacking [18]. It is worth mentioning that carbapenems are considered as a last line of drugs for the treatment of severe infections. Thus, the increasing frequency of Gramnegative bacteria producing extended spectrum enzymes able to inactivate carbapenems is a major public health concern [19].
Essential oils (EOs) are lipophilic, volatile compounds extracted from various parts of the plants (such as: leaves, roots, stem, flowers, buds, fruits, seeds, and woods), known for their antipathogen properties (such as: antibacterial, antifungal, antiviral, and insecticidal properties) [20]. They are secondary metabolites produced by the plants in order to counter fight the aggression of pests and predators, enhance seed dispersal or to attract the pollinators [20]. They represent a source of robust antimicrobial agent or reversal substances of drug-resistant strains against aggressive microbes [21]. Their wide spectrum of applications encompasses cosmetics (including perfumery and soap or even insect repellents), the beverage and food industry (such as flavoring agents, preservation additives, and disinfecting agents), agriculture (such as pesticides, fungicides or insecticides), as well as the drug industry (potent antimicrobial agents used in controlling healthcare related infections or as an alternative to medico-therapeutic techniques) [22].
Several studies reported EOs which possess direct antibacterial properties, as well as sensitizing or re-sensitizing actions on drug-resistant strains against pathogenic bacteria [23]. EOs are seen as agents with a great antimicrobial potential based on their natural origin. The high antibacterial effect of EOs was observed especially in members of Lamiaceae family and was attributed to the high content of thymol and carvacrol (monoterpenoid phenols, derivates of cymene) [24], even if, in some genera, the composition of essential oils can change, even considerably, depending on the different environmental conditions [25,26]. Data regarding the bioavailability of EOs in humans are extremely limited, with oral, pulmonary and dermal absorption being considered effective [20]. Due to their lipophilic structure, EOs can easily cross cell membranes, including the blood-brain barrier (with psychological effects in several central nervous system (CNS) diseases such as: anxiety, depression, insomnia, etc.) [27]. A dose-dependent selectivity of EOs was noticed, as well as a non-toxic effect against normal human cells has been demonstrated, although for the moment, limited data are available regarding they safety [20,28]. Among the commonly reported adverse effects of EOs are sensitization, irritation, photosensitization, dermatitis, neurotoxicity, organ toxicity, as well as endocrine imbalances [29]. Due to their low molecular weight, lipophilicity and protein binding ability, they can easily cross also the placenta, arriving to the fetal circulation and causing fetotoxicity [30][31][32]. Therefore, their safety profile in humans should be closely monitored and more intensively investigated, as what is currently known about the adverse effects of EOs is derived from non-systematic investigation and absence of spontaneous reporting systems, contrary to authorized medicinal products [29].
The purpose of the present article is to review the main pathogens classified by the WHO as critical in terms of current AMR, summarizing the most important aspects of resistance mechanisms to classical antibiotic therapy versus the latest findings regarding the efficacy of alternative essential oil therapy. Table 3 summarizes the effect EOs have on bacterial strains categorized as critical in terms of priority by WHO.    The nanoemultion was effective against the tested strains by inhibiting the drug efflux mechanism. Nanofromulation from Cleome viscosa L. proved to induce a broad-spectrum antimicrobial activity against the tested strains.

Melaleuca alternifolia (Maiden and Betche) and
Thymus vulgaris L. EOs were the most effective on the tested strains, the first EO being effective even at low concentrations. Melaleuca alternifolia (Maiden and Betche) MIC: 0.5-16 mcg/mL. Thymus vulgaris L. MIC: 1-16 mcg/mL. Both EOs presented also a good anti-biofilm activity, making them good candidates for infections caused by these pathogens even in association with classical antibiotics. The antimicrobial activity of tested EOs was superior when sodium chloride or caprylic acid was added. All tested EOs showed good antimicrobial activity against ESBL and non-ESBL-producing, as well as MDR and non-MDR E. coli strains. [59] Benameur  Chloroform extract induced the maximal antibacterial activity, with MBC: 2-5 mg/mL. Ampicillin MBC decreased from 10 mg/mL to 300-500 mcg/mL in the presence of Pimenta dioica (L.) Merr. Chloroform extracts and thus it induced a synergistic action. Eugenol was observed to be the major compound of the extract. EO induced a deformation in the bacterial cell membrane (stress and cellular damage).

Carbapenem-Resistant Acinetobacter baumannii
A. baumannii is a cause of serious healthcare associated infections. Some clinical mani-festations are pneumonia, bloodstream infections, infections of lower respiratory tract, urinary tract and wounds, skin infections, meningitis, osteomyelitis and endocarditis ( Figure 1) [72]. It has the ability to survive on abiotic surfaces and against disinfectant, which turns it into a "successful" nosocomial pathogen [73]. Plasticity of Acinetobacter spp. led to its rapid evolution in terms of developing resistance and turned it into a serious threat to hospitalized patients as treatment options are shrinking [74].

Carbapenem-Resistant Acinetobacter baumannii
A. baumannii is a cause of serious healthcare associated infections. Some clinical manifestations are pneumonia, bloodstream infections, infections of lower respiratory tract, urinary tract and wounds, skin infections, meningitis, osteomyelitis and endocarditis (Figure 1) [72]. It has the ability to survive on abiotic surfaces and against disinfectant, which turns it into a "successful" nosocomial pathogen [73]. Plasticity of Acinetobacter spp. led to its rapid evolution in terms of developing resistance and turned it into a serious threat to hospitalized patients as treatment options are shrinking [74]. Among "ESKAPE" pathogens (E. faecium, S. aureus, K. pneumoniae, A. baumanni, P. aeruginosa and E. cloacae), A. baumannii has been classified as being one of the most severe bacteria in terms of antimicrobial resistance (considered nowadays to be multidrug-resistant (MDR), extensively drug-resistant (XDR), and even pandrug-resistant strain (PDR)) as it shows resistance for almost all first-line antibiotics used for healthcare associated infections (problem that has emerged since the late '70s) [72,75].
Hospitalized patients have a higher risk of infection with A. baumanii due to its ability to penetrate through skin and respiratory devices [72]. The most common healthcare associated infection induced by A. baumannii is pneumonia, encountered mainly in patients in the intensive care unit as well as in immunocompromised ones [72,73,75].
A. baumannii strains can develop multiple mechanisms of antibiotic resistance. Currently, these strains are resistant to the broad-spectrum β-lactam antibiotics, carboxypenicillins, third generation of cephalosporins, and most recently to carbapenems [73]. Colistin and tigecycline have been reported to still be effective on MDR strains, alone or in combination, with some exceptions [73,76].
An important virulence factor in A. baumannii is the outer membrane protein (OmpA), which increases cell death by targeting mitochondria and the nucleus [72,75]. In addition, pure OMP38 induces apoptosis of human monocytes and epithelial cells [72,75]. Apoptosis of epithelial cells can reduce the surface of the mucosa and provide a way for bacteria to penetrate deeper into the tissues. Biofilms form on surfaces, and their matrix is composed of carbohydrates, nucleic acids, proteins, and other macromolecules [77]. The Among "ESKAPE" pathogens (E. faecium, S. aureus, K. pneumoniae, A. baumanni, P. aeruginosa and E. cloacae), A. baumannii has been classified as being one of the most severe bacteria in terms of antimicrobial resistance (considered nowadays to be multidrug-resistant (MDR), extensively drug-resistant (XDR), and even pandrug-resistant strain (PDR)) as it shows resistance for almost all first-line antibiotics used for healthcare associated infections (problem that has emerged since the late '70s) [72,75].
Hospitalized patients have a higher risk of infection with A. baumanii due to its ability to penetrate through skin and respiratory devices [72]. The most common healthcare associated infection induced by A. baumannii is pneumonia, encountered mainly in patients in the intensive care unit as well as in immunocompromised ones [72,73,75].
A. baumannii strains can develop multiple mechanisms of antibiotic resistance. Currently, these strains are resistant to the broad-spectrum β-lactam antibiotics, carboxypenicillins, third generation of cephalosporins, and most recently to carbapenems [73]. Colistin and tigecycline have been reported to still be effective on MDR strains, alone or in combination, with some exceptions [73,76].
An important virulence factor in A. baumannii is the outer membrane protein (OmpA), which increases cell death by targeting mitochondria and the nucleus [72,75]. In addition, pure OMP38 induces apoptosis of human monocytes and epithelial cells [72,75]. Apoptosis of epithelial cells can reduce the surface of the mucosa and provide a way for bacteria to penetrate deeper into the tissues. Biofilms form on surfaces, and their matrix is composed of carbohydrates, nucleic acids, proteins, and other macromolecules [77]. The biofilm protects bacteria from environmental damage by host responses, antibiotics, detergents and disinfectants. Therefore, biofilms contribute to prolonged and more severe bacterial effects [77]. Biofilm-producing A. baumanii strains have a higher survival rate than those without biofilm. These biofilm-producing strains showed a 10-13-day survival rate on dry, untouched surfaces in intensive care units compared to other Gram-negative bacteria. In addition, these strains are able to survive on hospital bed rails and in humid environments [77].
In a study conducted in 2019, the microscopic analysis of biofilm showed that the inhibition of biofilm formation is correlated with the duration of treatment and the dose of antibiotic administered. The inhibition was much more visible at longer administration and at a higher concentration [78].
Two articles were found regarding the effect of Origanum vulgare L. EO on some of WHO List of critical pathogens [34]. Detailed information is presented in Table 3. Origanum vulgare L. EO was found to induce destabilization and rupture of bacterial cell membrane, and thus, apoptosis, in A. baumannii-metallo-β-lactamase and carbapenemase producer strains (clinical isolates) by the study performed by Amaral SC et al., in 2020. Moreover, associated with polymyxin B, it induced a synergic activity (FICI: 0.18-0.37; checkerboard assay) with a 16 fold reduction in polymyxin's minimum inhibitory concentration (MIC) [34]. The Fractional Inhibitory Concentration Index (FICI) is a mathematical expression, used for describing the effect of antimicrobial agent combinations (e.g., additive, synergistic, antagonistic effects) [79]. It is defined as the MIC of drug A, in the presence of drug B, divided by the MIC of drug A alone (and vice versa). This concept gives an indication of the degree of drug interaction (e.g., synergy: FICI ≤ 0.5; no interaction: FICI > 0.5-4; antagonism: FICI > 4) [79].
Three studies were found regarding Mentha spp.'s antibacterial effect on carbapenemresistant A. baumannii strains [36,39,42]. In the article published by Rinaldi F and colleagues in 2020, the effect of two EOs (Thymus vulgaris L. and Syzygium aromaticum L.) chitosan coated nanoformulations was analyzed [38]. Both intranasal formulations manifested excellent antibacterial properties against carbapenem-resistant-A. baumannii and K. pneumoniae strains, with a MIC/MBC: 0.03% v/v. A remarkable antibacterial, concentration-dependent effect was observed in T. vulgaris L. against both types of bacteria. When the two main phytocompounds of the EOs, thymol and eugenol, were analyzed separately, they showed an unpredicted performance, suggesting the fact that the potent antimicrobial activity of T. vulgaris L. is attributed to the synergic actions of all active constituents of an EO, and not just one. S. aromaticum L. chitosan coated nanoformulations induced a bactericidal effect against A. baumannii-carbapenem-resistant strains after 2 h at a conc. of 0.125% v/v and after 6 h, at a conc. of 0.06% v/v, with absence of bacterial growth. In contrast, T. vulgaris L. nanoformulations induced a more rapid bactericidal effect, after 2 h incubation at a conc. of 0.06% v/v. Therefore, both EOs chitosan nanoformulations make them promising solutions against MDR bacterial strains of clinical concern, as for most of the available drugs, blood-brain barrier is the major limiting factor for drugs to reach subarachnoid space [38].

Carbapenem-Resistant Pseudomonas aeruginosa
Likewise, Pseudomonas aeruginosa is also an opportunistic pathogen, the most common bacteria responsible for healthcare associated infections ( Figure 1) and ventilator-associated pneumonia. It mainly affects cystic fibrosis patients and immunocompromised people and less often, healthy individuals [80]. Based on the fact that its genome is relatively large compared with other MDR strains, it has an enhanced capacity of coding regu-latory enzymes involved in the metabolism, transportation and efflux of organic substances, making it a bacterium with high adaptability and versatility to environmental changes [81]. Therefore, just as A. baumannii, it has developed the ability to resist most of the available antibiotics [82]. It is currently resistant to β-lactams, aminoglycosides and quinolones [80].
P. aeruginosa resistance to classical antibiotics has been classified and divided in three main types: intrinsic (by decreasing the outer membrane permeability, or over-expression of efflux pumps-which expel the antibiotic out of the cell or production of enzymes that inactivate the antibiotic), acquired (horizontal transfer of resistance genes = plasmids carrying genetic materials or mutations) and adaptive (formation of biofilm in the patient's lungs that prevents antibiotic penetration and which induce prolonged and recurrent infections) [80,83]. Moreover, the outer membrane of Gram-negative bacteria is another limiting factor for antibiotic penetration, as it acts as a selective barrier, planted with porins [80,82]. More specifically, the outer membrane of P. aeruginosa has a 12-100× decreased permeability compared with that of E. coli [84].
P. aeruginosa's resistance to broad-spectrum drugs such as carbapenems and cephalosporins is due to its ability to adapt by reducing the number of nonspecific porin proteins and replacing them with specific channels with low permeability to toxic chemicals [81]. Many carbapenem-resistant P. aeruginosa strains have been shown to be deficient in OprD porin (which is involved in antibiotic uptake and which contains the biding sites for carbapenems) [81].
The long presence of P. aeruginosa in clinical settings is attributed to the formation of biofilms on lung epithelial cells surfaces, through the production of DNA, proteins and exopolysaccharides [80,82]. These biofilms are characterized by effective cell-to-cell communication methods, known as quorum sensing. Three main quorum sensing systems (LasR, RhlI-RhlR, PQS-MvfR) are known contributors to the formation of mature and differentiated types of P. aeruginosa biofilms [80,82].
Modifications in the lipopolysaccharide (the central component of the Gram-negative bacterial membrane), production of bacterial enzymes (β-lactamases, metallo-β-lactamases, aminoglycoside modifying enzymes) as well as mutations are other mechanisms by which this pathogen can acquire resistance to classical antibiotics [86].
Various EOs are effective against carbapenem-resistant P. aeruginosa, as shown in Table 3. Oliva A et al., investigated, in the study published in 2018, the antibacterial effect of Melaleuca alternifolia L., tea tree oil (TTO) (used alone or in combination with classical antibiotics) against several MDR or PDR microorganisms (including carbapenem-resistant P. aeruginosa, A. baumannii, K. pneumoniae, as well as methicillin-resistant S. aureus), in both liquid and vapor phases. TTO expressed potent antibacterial activity, with MIC/MBCs of 0.25%/0.25% v/v for carbapenem-resistant A. baumannii, K. pneumoniae and of 1%/1% v/v for carbapenem-resistant P. aeruginosa. The MIC/MBC of TTO for methicillin-resistant Staphylococcus aureus (MRSA) was 0.5%/2% v/v. For all tested strains, an absence of bacterial growth was observed after 24 h incubation-period. TTO expressed a potent synergistic activity at sub-inhibitory concentrations with cefazolin (lowering the MIC from 32 to 1 mcg/mL), oxacillin (from 64 to 2 mcg/mL) and with amikacin against MRSA. Regarding the synergistic activity expressed with the tested Gram-negative strains, it showed a good bactericidal action with meropenem, amikacin and colistin. Therefore, the authors postulated that TTO could be taken into account as a possible non-conventional inhalation therapy for lung infections (e.g., caused by carbapenem-resistant A. baumannii) or other treatment regimens (used alone or in combination with classical antibiotics) against MDR and PDR strains [44]. K. pneumoniae is bacteria that commonly colonizes animal mucous membranes, from human to horses and swine. The environment represents a reservoir for human acquisition of this bacteria, given that K. pneumoniae is frequently found in water, sewage, soil, and plant surfaces [87]. There several strains of this pathogen: opportunistic, hypervirulent and MDR K. pneumoniae strains [88]. Opportunistic K. pneumoniae strains commonly colonize the gastrointestinal tract and might have a role to play in chronic diseases such as inflammatory bowel disease and colorectal cancer (Figure 2) [89]. In vulnerable patients, K. pneumoniae can also cause extraintestinal infections, such as urinary tract infection, pneumonia, bloodstream infections, wound or surgical site infections and sepsis ( Figure 2) [90]. Opportunistic strains are generally acting in healthcare settings, affecting newborns, elderly, immunocompromised and hospitalized patients [90]. Other strains of K. pneumoniae are hypervirulent, infecting healthy people in community settings. They cause severe infections, such as pyogenic liver abscess, endophthalmitis, and meningitis [91]. bactericidal action with meropenem, amikacin and colistin. Therefore, the authors postulated that TTO could be taken into account as a possible non-conventional inhalation therapy for lung infections (e.g., caused by carbapenem-resistant A. baumannii) or other treatment regimens (used alone or in combination with classical antibiotics) against MDR and PDR strains [44].

Carbapenem-Resistant and ESBL-Producing Klebsiella pneumoniae
K. pneumoniae is bacteria that commonly colonizes animal mucous membranes, from human to horses and swine. The environment represents a reservoir for human acquisition of this bacteria, given that K. pneumoniae is frequently found in water, sewage, soil, and plant surfaces [87]. There several strains of this pathogen: opportunistic, hypervirulent and MDR K. pneumoniae strains [88]. Opportunistic K. pneumoniae strains commonly colonize the gastrointestinal tract and might have a role to play in chronic diseases such as inflammatory bowel disease and colorectal cancer (Figure 2) [89]. In vulnerable patients, K. pneumoniae can also cause extraintestinal infections, such as urinary tract infection, pneumonia, bloodstream infections, wound or surgical site infections and sepsis ( Figure 2) [90]. Opportunistic strains are generally acting in healthcare settings, affecting newborns, elderly, immunocompromised and hospitalized patients [90]. Other strains of K. pneumoniae are hypervirulent, infecting healthy people in community settings. They cause severe infections, such as pyogenic liver abscess, endophthalmitis, and meningitis [91]. Regarding the virulence of K. pneumoniae, so far four pathogenicity factors have been identified: pili, capsule, lipopolysaccharides (LPS), and siderophore [92,93]. These factors are chromosomally encoded and represent the basic requirements for establishing opportunistic infections. Type 1 and type 3 pili promote bacterial adhesion to epithelial, immune cells, and abiotic surfaces [91]. LPS modifications make it difficult for K. pneumoniae to be recognized by the host cell [94]. K. pneumoniae needs to acquire iron from the environment in order to thrive during infection. This is accomplished through the secretion of Regarding the virulence of K. pneumoniae, so far four pathogenicity factors have been identified: pili, capsule, lipopolysaccharides (LPS), and siderophore [92,93]. These factors are chromosomally encoded and represent the basic requirements for establishing opportunistic infections. Type 1 and type 3 pili promote bacterial adhesion to epithelial, immune cells, and abiotic surfaces [91]. LPS modifications make it difficult for K. pneumoniae to be recognized by the host cell [94]. K. pneumoniae needs to acquire iron from the environment in order to thrive during infection. This is accomplished through the secretion of siderophores-molecules with a higher affinity for iron than host proteins-such as enterobactin, yersiniabactin, salmochelin, and aerobactin [95]. Elevated virulence through capsule modifications is obvious within the hypervirulent strains, associated with hyper-mucoviscosity. This is due to increased capsular polysaccharide production, mediated by RmpA and/or RmpA2 [91]. Moreover, K. pneumoniae is capable of forming biofilms-thick outer layers that favors bacterial attachment to surfaces. This asset also gives protection against ABs penetration, thus reducing their effects [96].
In terms of AMR, K. pneumoniae is naturally resistant to ampicillin due to the presence of the SHV-1 penicillinase in its chromosome. Resistance to other ABs occasionally occurs through chromosomal mutations, generally via horizontal gene transfer [97]. K. pneumoniae is increasingly developing resistance genes for aminoglycosides, quinolones, β-lactams, carbapenems, polymyxin and tigecycline. Aminoglycoside resistance is related to modification of cell permeability and the presence of aminoglycoside-modifying enzymes genes [98,99]. It has been reported that KpnO pore proteins, as well as KpnEF efflux pump systems and AcrAB-TolC, are directly involved in blocking aminoglycosides [98,99]. The primary Quinolone resistance mechanism is represented by the mutation of gyrA-gyr B subunit of DNA gyrase and parC-parE subunit of topoisomerase IV. Changes in cell permeability are also involved, including overexpression of the multidrug efflux pump gene acrAB, non-alteration of kdeA and OmpK36 deficiency [100][101][102][103][104]. The main mechanism of polymyxin resistance is target modification by chromosomal modifications. K. pneumoniae is able to change the structure of lipopolysaccharides in its membrane, thus affecting polymyxin binding through negative ion reduction [105]. Tigecycline resistance is acquired through modifications of the 30S and 16S ribosomal unit targets, along with changes in cell permeability, overexpression of efflux pumps AcrAB-TolC and OqxAB and changes in the expression levels of their regulators (RamA, RamR, RarA, and AcrR) [106].
K. pneumoniae encodes class A β-lactamase enzyme SHV in the core gene blaSH, which often suffers mutations and is routinely getting integrated into mobile genetic elements [107]. This accessory AMR gene is then horizontally transferred to other strains, resulting in ESBL activity and conferring resistance to third-generation cephalosporins and even carbapenems. Thus, K. pneumoniae is a crucial entry point of AMR genes into the Enterobacterales family [107]. In terms of human morbidity and mortality, carbapenem-resistant K. pneumoniae represents the fastest growing AMR threat in Europe [108]. Carbapenemresistance is mediated by two primary mechanisms. The first one involves β-lactamases production and reduction in membrane permeability. β-lactamases, such as AmpC cephalosporinase (DHA-1 or CMY-2) or ESBL (CTX-M-2), are able to hydrolyze cephalosporins [109]. The second mechanism is mediated by the production of a β-lactamase capable of hydrolyzing most β-lactams antibiotics including carbapenems. These β-lactamases are called carbapenamases and can be divided into: K. pneumoniae carbapenemase (KPC), metallo-β-lactamases (VIM, IMP, NDM), and OXA-48 type enzymes [110]. Convergence of carbapenem resistance with hypervirulence results in super virulent strains, associated with high mortality rates [110]. In 2018, a first report indicated that carbapenem-resistant K. pneumoniae has acquired a virulence plasmid from hypervirulent K. pneumoniae strains [111].

Carbapenem-Resistant and ESBL-Producing Escherichia coli
E. coli is not only the most prevalent commensal inhabitant in gastrointestinal tracts, but it is also one of the most important pathogens [112]. It can cause both intestinal pathologies (various forms of enteritis diarrhea, including hemolytic and uremic syndrome) and extraintestinal pathologies (urinary tract infections, diverse intraabdominal, pulmonary, skin and soft tissue infections, newborn meningitis and bacteremia) (Figure 2) [113]. E. coli is highly used in biological sciences, medicine, and industry, being the most popular microorganism in the field or recombinant DNA technology [114]. It is an important host organism in biotechnology, due to ease of handling, availability of the complete genome sequence, and its ability to grow under both aerobic and anaerobic conditions [114].
So far, eight E. coli pathotypes causing disease in humans have been described, six causing intestinal and two extraintestinal infections. The six intestinal pathotypes are: enteropathogenic, enterohaemorrhagic, enterotoxigenic, enteroaggregative, enteroinvasive and diffusely adherent E. coli. Extraintestinal pathogenic E. coli is divided into uropathogenic and meningitis-associated E. coli [115]. Among E. coli virulence, the biofilm formation contributes to the occurrence of infections and makes their eradication difficult. For instance, catheter-associated urinary tract infections-one of the most healthcare associated infections-are associated with the biofilm formed by E. coli on catheters [116]. Porins have also an important role to play in AMR, especially OmpA, OmpC and OmpF [117].
E. coli is very effective in acquiring resistance genes through horizontal gene transfer. Some of these genes are coding 16S rRNA methylases, inactivating aminoglycosides or plasmid-mediated quinolone resistance genes, conferring resistance to fluoroquinolones [118]. Other genes encode for ESBL, thus conferring resistance to broadspectrum cephalosporins. This includes the ubiquitous class A ESBL enzymes, as well as class C β-lactamases-AmpC-type enzymes (CMY, DHA, ACC)-responsible for high-level resistance to cephalosporins [119].
Last but not least, a growing number of E. coli strains are able to produce carbapenamases such as class A KPC, class B metallo-β-lactamases (NDM, VIM and IMP) and class D OXA-48 [120]. These enzymes are encoded by blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA genes present in both the chromosome and the plasmid [121]. Carbapenamases are associated with difficulties in detection and treatment failure, especially OXA-48-like enzyme [122]. Resistance to carbapenems is also mediated by mutations in membrane porins (especially OmpC and OmpF) and overexpression of efflux pumps [19].
The studies we found regarding the antimicrobial and antibiofilm activity of EOs on carbapenem-resistant and ESBL-producing Enterobacterales strains are presented in Table 3. The EOs were tested either as food additives (Thymbra capitata (L.) Cav.EO was proposed as food preservative for meat by Aouadhi C et al., 2022 [50]; Artemisia herba-alba Asso. and Thymus algeriensis Boiss. and Reut. EOs were proposed as natural antibacterial agents for milk shelf life as they showed a good antimicrobial activity against ESBL-producing Enterobacterales strains isolated from raw milk in the study performed by Sara M et al., 2021 [70]) or even substitutes for classical antibiotics in animal sector (poultry) in the study performed by Shrivastav A et al., 2019 on Syzygium aromaticum L. EO [55].
The studies performed by Tebrun W et al., 2020 and Motola G et al., 2020 analyzed the efficacy of several EOs as possible disinfection methods on eggshell samples [52,54]. In the study performed by Motola G et al., in 2020, from the six methods that were tested, only five of them (formaldehyde gassing; hydrogen peroxide + alcohol spray; EOs spray; peracetic acid foam and low energetic electron radiation) managed to decrease or completely eliminate the ESBL-producing E. coli strains. In contrast, the EOs spray that was used as cold fog could only partially reach the expected efficacy point [52].
EOs nanoformulations were also tested against different MDR strains. The study performed by Krishnamoorthy R et al., 2018 highlighted the antimicrobial activity of Cleome viscosa L. essential oil nanoemulsion (droplet size: 7 nm and 1:3 (v/v) oil: surfactant ratio) against ESBL-producing E. coli strains, K. pneumoniae, and P. aeruginosa, as well as against MRSA. The EO nanoformulation inhibited the drug efflux mechanism by damaging the cell membranes and walls of the tested strains (the active phytocompounds of the EO being: β-sitosterol, demecolcine, campesterol, and heneicosyl formate) [53].
Origanum onites L. EO was proposed as a possible alternative to synthetic antibacterial drugs for its antibacterial activity on ESBL-producing E. coli strains, after testing also its toxic effects and risks of irritation at therapeutic doses in humans, by the study performed by Kaskatepe B et al., 2017 [56], although there are also other species of the genus Origanum with these properties [123].
Vasquez NM et al. reported, in 2020, the antibiofilm activity of eucalyptol (the main constituent of rosemary volatile oil) on ESBL-producing E. coli strains isolated from urinary samples of adult patients [71]. Its bactericidal activity of eucalyptol had been already reported by the same team on E. coli ATCC 35218 strains in 2013 (MIC: 0.8% (v/v)) [124]. In the study, 1,8-cineole presented a concentration-dependent and a time-dependent antibiofilm activity against the biofilms that were pre-formed [71]. After only 1 h of treatment with 1% (v/v) a 3-log reduction in the viable biofilm cells was noticed, suggesting an important bactericidal activity [71].
Both studies performed by de Souza et al., 2021 and Dhara L et al., 2020 evaluated the in vitro as well as in vivo effects (animal model) of carvacrol and cinnamaldehyde and eugenol, respectively, on different carbapenem-resistant or ESBL-producing Enterobacterales strains. The in vitro antimicrobial activity of carvacrol against carbapenem-resistant K. pneumoniae was observed in all isolates, with a bactericidal activity observed within 4 h [62]. The in vivo antibacterial effect was showed on a mouse model of carbapenemresistant K. pneumoniae infection. Carvacrol treatment increased survival and significantly decreased the bacterial load in the peritoneal lavage of the mouse. Moreover, the animals under carvacrol treatment expressed a decrease in number of leukocytes and an increase in number of platelets, but the values were within the normal range, when compared with the control group of animals [62].
The study performed by Dhara L. et al., in 2020, described the antibacterial activity of eugenol and cinnamaldehyde against ESBL producing and quinolone resistant Enterobacterales. Moreover, the in vivo toxicity of these EOs was investigated, in order to observe their pharmacological potential as future therapeutical solutions. Both EOs manifested good antimicrobial activity against the tested strains (data presented in Table 3), as well as good safety profiles (toxicological and behavioral effects), evaluated through a 14-day study on hematological and toxicological analyses after oral ingestion of eugenol (7.34-70 mg/kg) and cinnamaldehyde (0.91-10 mg/kg) [63].
The same team of Dhara L. and colleagues, investigated in the study published in 2020 the antimicrobial effect of cinnamaldehyde used alone or in combination with cefotaxime/ciprofloxacin. Synergism of cinnamaldehyde with cefotaxime was observed. Moreover, cinnamaldehyde managed to decrease MIC of cefotaxime and ciprofloxacin up to 1024×, with a bactericidal and synergistic effect observed after 24 h [64].
Qian W. and colleagues showed, in the study published in 2019, the antimicrobial effect of eugenol, as well as its effect on biofilm formation and biofilm-associated gene expression of carbapenem-resistant K. pneumoniae strains. Eugenol managed to deteriorate cell membrane by decreasing ATP concentration, reducing its pH, increasing hyperpolarization and by stimulating its permeability [68].

Materials and Methods
The first step in the elaboration of the present review was the selection of the topic. Based on the current issues of AMR and the WHO list of bacteria that require imperative new solutions of treatment, three researchers (V.B., C.D and D.M.) conducted a search in the scientific literature in order to gather more data regarding this topic. They noticed that updated reviews on the efficacy of EOs on different MDR strains were lacking; therefore, they decided to conduct a review on this subject. The aim was to identify the latest findings regarding the efficacy of EOs on the WHO list of critical pathogens (also known as Priority 1 pathogens). A working team was established with members having experience in the area of interest: clinical laboratory, microbiology, botany, phytotherapy and clinical pharmacology.
From March 2022 to end of April 2022, authors conducted independent research on 2 electronic databases (PubMED and Web of Science) in order to identify original articles describing the activity of EOs on each critical pathogen mentioned in the WHO's list ( Figure 3). The keywords used to perform this research were the following: Name of WHO critical pathogen AND antibiotic resistance AND essential oils (e.g., A. baumannii AND carbapenem-resistant AND essential oils). Articles that were published starting from January 2016 were included (in vitro as well as in vivo studies). Inclusion criteria: WHO Priority 1 pathogens; the specific type of resistance mentioned by WHO; articles discussing the antibacterial and/or antibiofilm efficacy of a minimum EO; from 2016 to 2022. A template (same table head as in Table 3) was built by the working team for data extraction.
After each independent researcher extracted the required data, a face-to-face meeting was conducted in order to discuss the highlights of each article. Data validation and disagreements were solved by a researcher with expertise in microbiology (D.M.).
The mechanism of developing resistance for each pathogen was documented by a B.B. and a graphic representation of the main clinical diseases induced by each bacterium was made by A.L., using BioRender.com (accessed on 1 June 2022).

Conclusions
As the golden era of classical ATB seems to be over due to the occurrence of MDR strains, medical practitioners must be opened to new approaches in treating infections. In this regard, healthcare associations need to come up with innovative measures to prevent and slowdown of AMR and establish a global antimicrobial guide that completely tackles this global health issue. At the same time, the entire scientific community needs to continuously work on investigating alternative solutions to fight AMR. There are some directions that are currently under exploration, such as identifying new compounds with different mechanisms of actions; developing alternative administration routes for the existing compounds; and working on the formulations through adjusting the adjuvant substances. However, one key tool in fighting AB resistance might actually lie in the thorough understanding of the underlying mechanism of AMR. Knowledge about how and when After each independent researcher extracted the required data, a face-to-face meeting was conducted in order to discuss the highlights of each article. Data validation and disagreements were solved by a researcher with expertise in microbiology (D.M.).
The mechanism of developing resistance for each pathogen was documented by a B.B. and a graphic representation of the main clinical diseases induced by each bacterium was made by A.L., using BioRender.com (accessed on 1 June 2022).

Conclusions
As the golden era of classical ATB seems to be over due to the occurrence of MDR strains, medical practitioners must be opened to new approaches in treating infections. In this regard, healthcare associations need to come up with innovative measures to prevent and slowdown of AMR and establish a global antimicrobial guide that completely tackles this global health issue. At the same time, the entire scientific community needs to conti-nuously work on investigating alternative solutions to fight AMR. There are some directions that are currently under exploration, such as identifying new compounds with different mechanisms of actions; developing alternative administration routes for the existing compounds; and working on the formulations through adjusting the adjuvant substances. However, one key tool in fighting AB resistance might actually lie in the thorough understanding of the underlying mechanism of AMR. Knowledge about how and when resistance occurs can help minimize the emergence of MDR pathogens, since new ABs are developed at a slow pace. Thus, the real challenge nowadays is how to make best use of the available assets, including technology, knowledge, and actives such as classical ABs and EOs. Little is known regarding the efficacy and safety of EOs in humans, given that most of the existent research being conducted in vitro. Consequently, much more research is needed in order to have a clear picture on the benefits, as well as the risks of using EOs as alternative to the current, classical antimicrobial therapy, in humans. Moreover, finding efficacious and safe synergistic drug combinations could be another solution for the current challenges of AMR. Funding: This research received no external funding.

Data Availability Statement:
All the data is presented in the present manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.